Method for aiding implantation or decreasing miscarriage rate

ABSTRACT

The invention provides a protocol leading to improved embryo implantation rates and/or decreased miscarriage rates in which hCG or LH, or a bio-analogue thereof, is administered during the follicular phase.

FIELD OF INVENTION

The invention relates to the field of in vivo and in vitro assistedreproduction technologies (ART), specifically controlled ovarianhyperstimulation (COH) using gonadotropins.

BACKGROUND OF THE INVENTION

Treatment of infertility by assisted reproduction technologies (ART)such as in vitro fertilisation (IVF) or IVF in conjunction withintracytoplasmic sperm injection. (IVF/ICSI) and embryo transfer (ET)requires controlled ovarian hyperstimulation (COH) to increase thenumber of oocytes¹. Standard regimens² for COH include a down-regulationphase in which endogenous luteinising hormone (LH) is suppressed byadministration of a gonadotropin releasing hormone (GnRH) agonistfollowed by a stimulation phase in which follicular development(folliculogenesis) is induced by daily administration of folliclestimulating hormone (FSH), usually at about 150 IU/day. Other moleculeshaving FSH activity may also be used. Alternatively stimulation isstarted after spontaneous or induced menstruation while preventing theoccurrence of an LH surge by administration of a GnRH-antagonist,usually starting on about day 6 or 7 of FSH administration. Insuperovulation protocols for ART, multiple follicular development is thedesired aim. When there are at least 3 follicles >16 mm (one of 18 mm),a single bolus of hCG (5-10,000 IU) is given to trigger ovulation.Oocyte recovery is timed for 36-38 hours after the hCG injection.

The rationale for the use of GnRH agonists and antagonists in thiscontext is the prevention of an untimely LH surge that would causepremature ovulation and follicle luteinisation³. GnRH agonist regimenshave become the accepted norm in the clinic. It has been found that longregimens (i.e., those started in the midluteal phase of the cyclepreceding ovulation induction, or before) are associated with easierpatient scheduling, greater follicle yield, and overall better clinicalresults.⁴ The use of GnRH antagonists is relatively new to the clinic,but is expected to show similar benefits, with the added advantage of ashorter treatment regimen.

The prolonged administration of GnRH agonists or the administration ofGnRH antagonists results in profound suppression of endogenous LH. Thissituation, while not incompatible with follicle development, does notmimic the natural cycle. In the natural cycle, LH levels show a gradualincrease several days before the peak at midcycle.

Several groups have explored the role of LH and chorionic gonadotropin(CG) in ovulation induction and ART. As is well known and recognised inthe art, techniques or methods of ovulation induction (OI) are distinctfrom methods of COH, although both may involve the administration ofFSH.

Hillier et al. have demonstrated that very low levels of LH suffice forfolliculogenesis⁵.

Esposito et al. have studied the role of endogenous LH in ART cyclesstimulated with rFSH. They conclude that follicular fluid estradiollevels, oocyte yield, and fertilisation improve when serum LHconcentrations are higher than 0.5-1.0 IU/L⁶.

WO 00/67778 (Applied Research Systems) discloses the use of LH duringthe stimulatory phase for inducing folliculogenesis in ovulationinduction, particularly to encourage the development of a singledominant follicle.

The European Recombinant Human LH Study Group reports thatadministration of rhLH (75 and 225 IU/day) for supporting rhFSH-inducedfollicular development in hypogonadotropic hypogonadal women increasesthe number and size of follicles⁷, with respect to a control groupreceiving only rhFSH.

Filicori et al. have investigated the role of low doses of hCG, as asurrogate for LH, in controlled ovarian hyperstimulation (Filicori, etal., J. Clin. Endocrin. Metab., 84, 1999, 2659-2663). hCG administration(50 IU hCG/day) was started synchronously with FSH administration andwas continued on a daily basis until ovulation was triggered with abolus of hCG. The numbers of small (<10 mm), medium (10-14 mm) and large(>14 mm) follicles were comparable between a group receiving hCG and acontrol group receiving FSH alone, however, the cumulative dose of FSHand the duration of FSH stimulation were reduced in the hCG treatedgroup.

Messinis et al. report ovulation induction in anovulatory women (WHOgroup I) using a regimen that uses daily doses of hMG (75 IU each of FSHand LH) during the stimulatory phase and single or multiple doses of hCGduring the luteal phase. The pregnancy rate was found to besignificantly increased in patients receiving multiple hCG doses duringthe luteal phase as compared to a control group that received only asingle ovulation inducing/triggering dose of hCG⁸.

Proper follicular development is of course essential for successful ARTmethods. However, there are some cases in which ovulation andfertilisation are achieved, and yet improper implantation of the embryoprevents pregnancy. In other cases, spontaneous abortion (miscarriage)occurs during the first trimester. Both these problems may be associatedwith conditions in the endometrium, which is quite sensitive to hormonelevels. Thus, it can be seen that even once follicular development,ovulation and fertilisation have occurred there is no guarantee of asuccessful pregnancy and problems with implantation and earlymiscarriage are often encountered.

In some patients, tendency to abort or failure to implant may eventuallybe overcome, but to do so requires repeated ART cycles, with consequentnegative physiological and psychological effects on the patient. Inother patients, these problems represent an essentially permanentstumbling block to pregnancy.

Methods for increasing implantation rates and decreasing earlymiscarriage rates, particularly in conjunction with COH, are thus highlydesirable.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an improved method ofadministration of gonadotropins for COH, leading to improvedimplantation rates and decreased miscarriage rates.

It is a further object of the invention to provide a method forincreasing pregnancy rates, e.g. by improving implantation rates and/ordecreasing miscarriage rates, in patients who are not undergoing COH,for example, in patients following a natural ovulatory cycle or inpatients undergoing ovulation induction.

In a first aspect, the invention provides a use of human chorionicgonadotropin (hCG) or an analogue thereof, for the manufacture of amedicament for encouraging implantation and/or decreasing miscarriagerates of an embryo in a human patient, wherein said medicament isadministered before ovulation or ovulation triggering.

In a second aspect, the invention provides a use of human chorionicgonadotropin (hCG) or an analogue thereof, for encouraging implantationand/or decreasing miscarriage rates of an embryo in a human patient,wherein said hCG or an analogue thereof is administered before ovulationor ovulation triggering.

In a third aspect, the invention provides a use of human chorionicgonadotropin (hCG), or an analogue thereof, for the manufacture of amedicament for use in conjunction with controlled ovarianhyperstimulation (COH) in human patients using FSH, or an analoguethereof, for aiding implantation and/or decreasing miscarriage rates,wherein the medicament is to be administered starting before the 10^(th)day after commencing FSH treatment.

In a fourth aspect, the invention provides a use of human chorionicgonadotropin (hCG), or an analogue thereof, in conjunction withcontrolled ovarian hyperstimulation (COH) in human patients using FSH,or an analogue thereof, for aiding implantation and/or decreasingmiscarriage rates, wherein the hCG or an analogue thereof is to beadministered starting before the 10^(th) day after commencing FSHtreatment.

In a fifth aspect, the invention provides a pharmaceutical compositionfor use in aiding implantation of an embryo and/or decreasingmiscarriage rates, optionally and preferably in conjunction with COH,comprising 25-1000 IU hCG, or an analogue thereof, per dosage.

In a sixth aspect, the invention provides a use of human luteinisinghormone (hLH), or an analogue thereof, for the manufacture of amedicament for use in conjunction with controlled ovarianhyperstimulation (COH) in human patients using FSH, or an analoguethereof, for aiding implantation and/or decreasing miscarriage rates,wherein the medicament is to be administered starting before the 10thday after commencing. FSH treatment, preferably between the 3^(rd) andthe 10^(th) day after commencing FSH treatment.

In a seventh aspect, the invention provides a use of human luteinisinghormone (hLH), or an analogue thereof, in conjunction with controlledovarian hyperstimulation (COH) in human patients using FSH, or ananalogue thereof, for aiding implantation and/or decreasing miscarriagerates, wherein the LH is to be administered starting before the 10th dayafter commencing FSH treatment, preferably between the 3^(rd) and the10^(th) day after commencing FSH treatment.

In a further aspect, the invention provides a use of LH or an analoguethereof, for the manufacture of a medicament for encouragingimplantation and/or decreasing miscarriage rates of an embryo in a humanpatient, wherein said medicament is administered before ovulation orovulation triggering.

In a yet further aspect, the invention provides a use of LH or ananalogue thereof, for encouraging implantation and/or decreasingmiscarriage rates of an embryo in a human patient, wherein said LH or ananalogue thereof is administered before ovulation or ovulationtriggering.

In a further aspect, the invention provides a pharmaceutical compositionfor use in aiding implantation of an embryo and/or decreasingmiscarriage rates, optionally and preferably in conjunction with COH,comprising 150-1000 IU LH, or an analogue thereof, per dosage.

A further aspect of the invention provides hCG or an analogue thereof orLH or an analogue thereof for use in conjunction with controlled ovarianhyperstimulation (COH) in human patients using FSH, or an analoguethereof, for aiding implantation and/or decreasing miscarriage rates,wherein the hCG or an analogue thereof, or the LH, or an analoguethereof is to be administered starting before the 10^(th) day aftercommencing FSH treatment.

In an alternative embodiment, the invention provides hCG or an analoguethereof or LH or an analogue thereof for encouraging implantation and/ordecreasing miscarriage rates of an embryo in a human patient, whereinsaid hCG or LH or the analogues thereof are administered beforeovulation or ovulation triggering.

In a yet further aspect, the present invention provides a method ofencouraging implantation and/or decreasing miscarriage rates in apatient, which method is used in conjunction with controlled ovarianhyperstimulation using FSH, or an analogue thereof, which methodcomprises the administration to the patient of hCG or an analoguethereof, or LH or an analogue thereof, wherein said administrationstarts before the 10^(th) day after commencing FSH treatment.

The present invention also provides a method of encouraging implantationand/or decreasing miscarriage rates in a patient, which method comprisesadministering to the patient hCG or an analogue thereof, or LH or ananalogue thereof, wherein said hCG or LH or the analogues thereof areadministered before ovulation or ovulation triggering.

In a further aspect, the invention provides a kit for use in COHcomprising 12 or more, preferably 14 or more daily doses of FSH,preferably about 75-200 IU FSH/day, more preferably about 150 IUFSH/day, and 4 to 8, 5 to 8 or 6 to 8 daily doses of hCG, for example 4,5, 6, 7 or 8 daily doses of hCG, preferably about 25-1000 IU hCG/day,more preferably about 50-100 IU hCG/day.

In a further aspect, the invention provides a kit for aidingimplantation of an embryo and/or decreasing miscarriage rates, the kitcomprising 4 to 8, 5 to 8 or 6 to 8-daily doses of hCG, for example 4,5, 6, 7 or 8 daily doses of hCG, at about 25-1000 IU hCG/day, preferablyat about 50-100 IU hCG/day. Such kits may or may not be used inconjunction with COH.

Preferably the kits and pharmaceutical compositions of the invention aredesigned for use in the methods and uses of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Human chorionic gonadotropin (hCG) is a heterodimeric glycosylatedpeptide hormone which is produced by the placenta during pregnancy. Itappears in serum shortly after fertilisation, and acts to maintain thecorpus luteum after LH secretion decreases, supporting continuedsecretion of oestrogens and progesterone and preventing menstruation.Because it is only present at significant levels in the pregnant female,it is not thought to play a significant role in the natural ovulatorycycle. It is known that receptors for LH/hCG exist in the gonads, theuterus, fallopian tubes, placenta and in endometrial and myometrialcells⁹. hCG has the longest half life of the gonadotropins¹⁰.

The inventors have unexpectedly discovered that administration of hCG oran analogue at low doses during the stimulatory phase of ART cycles hasa beneficial effect on pregnancy rates, e.g. by aiding or increasingimplantation and/or by decreasing miscarriage rates. The expression “lowdoses” encompasses doses less than the dose conventionally used in aparticular patient to trigger follicle maturation, i.e. final follicularmaturation, just prior to ovulation (“the follicle (or ovulation)triggering dose”). The follicle/ovulation triggering dose of hCG(usually in the range of 5,000-10,000-IU hCG), will vary on apatient-to-patient basis.

As can be seen from the discussion of standard COH regimens above, theadministration of this “high dose” or ovulation triggering dose of hCGis often referred to in the art as the ovulation triggering step, oocytematuration step or ovulation stimulation step. Such an ovulationstimulation or triggering step or oocyte maturation step involving theadministration of a high dose of hCG is however only carried out onceadequate follicular development has been achieved during the stimulatoryphase of the COH regime, i.e. the phase involving the administration ofFSH or an analogue thereof to induce folliculogenesis. A majordifference between the methods and uses of the present invention and theprior art regimens is that hCG is administered during the stimulatoryphase, i.e. before adequate follicular development has been achieved andbefore ovulation occurs or is triggered, and is administered at dosesbelow that which induce oocyte maturation and ovulation stimulation,i.e. at doses below the ovulation triggering dose. Such regimens of thepresent invention give rise to surprising advantages in terms ofimplantation and miscarriage. Thus, in accordance with the presentinvention the low doses of hCG are administered in conjunction with COHregimes during the stimulatory phase before adequate folliculardevelopment has occurred and before the final high ovulation triggeringhCG dose is administered to trigger oocyte maturation and ovulation.

In aspects of the invention where the patients are not undergoing COHlow doses of hCG are also administered during the phase offolliculogenesis, and before adequate follicular development has beenachieved. Again these doses of hCG are below the ovulation triggeringdose. As indicated above, such patients may have a natural ovulatorycycle, in which case the eventual administration of an ovulationtriggering dose of hCG will not be necessary. However, some of thepatients not undergoing COH which can be treated by the administrationof low doses of hCG in accordance with the methods of the invention maynot have a natural ovulatory cycle (e.g. those undergoing OI), in whichcase the administration of an ovulation triggering dose of hCG can becarried out once adequate follicular development is judged to have beenachieved.

As mentioned above, the invention also provides the administration of LHrather than hCG in the methods and uses of the invention. Theadministration of LH is also carried out during the phases of the cycleand follicular development as described herein for hCG.

When hCG is used in the aspects of the invention described herein, thedosage should be in the range of 25-4000 IU hCG/day, preferably 25-1000IU hCG/day, more preferably 30-1000 or 30-500 IU hCG/day, andparticularly preferably 50-100 IU or 75-125 or 75-100 IU hCG/day or 75or 100 to 500 or 75 or 100 to 1000 IU/day. Such doses are below theovulation triggering dose and, as described above, are also referred toherein as “low doses” of hCG. If an hCG analogue is used, the equivalentto these hCG doses should be administered.

As indicated above pharmaceutical compositions or kits which may containsuch doses of hCG (or doses of LH as described elsewhere herein) for usein the methods and uses of the invention, are also provided.

In aspects of the invention where hCG (or an analogue thereof) is usedin conjunction with COH using FSH, or an analogue thereof,administration of hCG (or analogue) should begin before the 10^(th) dayafter starting treatment with FSH, more preferably before the 9^(th) dayafter starting treatment with FSH. Administration of hCG shouldpreferably not be started until at least 3 days after beginning FSHtreatment, for example between the 3rd, and 10th day after starting FSHtreatment, more preferably not until at least 5 or 6 days afterbeginning FSH treatment. Particularly preferably, administration of hCGshould be started on or about the 7^(th) or 8^(th) day aftercommencement of FSH treatment. Thus, hCG is administered during thefollicular phase and preferred timepoints for administration of hCG areat or about the mid-follicular stage of the cycle, i.e. at least 5, 6, 7or 8 days after commencement of FSH treatment.

The administration of hCG in accordance with the present invention maybe a single, bolus, in which case it should preferably take place on orabout the 7^(th) or 8^(th) day after FSH treatment is started, and thedosage should preferably comprise 100-1000 IU hCG, more preferably100-500 IU hCG or 150-600 IU hCG, and particularly preferably about 250IU hCG. Administration as a single bolus has the advantage ofconvenience for both the practitioner and the patient.

Alternatively, administration of hCG in accordance with the presentinvention may be carried out on a daily basis until follicle maturationis triggered or ovulation is induced/triggered with the conventionalbolus of hCG. For daily administration, the dosage should be in therange of 25-4000 IU hCG/day, preferably 25-1000 IU hCG/day, morepreferably 30-1000 or 30-500 IU hCG/day, most preferably 50-100 IU or75-125 IU hCG/day or 75-100 or 75 or 100 to 500 or 75 or 100 to 1000IU/day. A daily regimen that starts on the 7^(th) day after commencingFSH treatment, and which uses 50-100 IU hCG/day has been found to beparticularly effective. It is also possible to administer hCG on a lessfrequent basis, for example every two, three, or four days, preferablyevery two days, until ovulation is triggered. In such regimen doses suchas those outlined above may be used, although a dose of 50-200 IU hCG ispreferred.

From the above discussion it will be apparent that the hCG (or LH) usedin accordance with the present invention is administered starting beforeovulation occurs or before ovulation is triggered, e.g. by the ovulationtriggering dose of hCG, and is continued until ovulation occurs or istriggered. In all the regimens of the invention, the administration ofhCG (or LH) may, if desired, be continued after ovulation if this isthought to be of benefit to the patient.

The timing at which ovulation may be triggered by administration of the“follicle/ovulation triggering dose” of hCG will be well known to aperson skilled in ART regimens and can be determined accordingly. Ingeneral ovulation is triggered when follicle development is consideredadequate for the type of regimen being used. The level of follicledevelopment is generally determined by measuring the size of thefollicles (e.g. by ultrasound) and the serum oestradiol (E₂) level ofthe patient. If the regimen in question is COH then because the aim ofthese methods is multiple follicular development to produce an increasednumber of mature follicles/oocytes, which are generally fertilised invitro and reintroduced to the patient, then the timing of the ovulationtriggering is likely to be slightly different than if the regimen inquestion is ovulation induction in which the aim is to produce one, orat the most two, mature follicles which are ovulated and fertilised invivo. For example in embodiments involving COH regimens ovulation may betriggered with 5000 to 10000, e.g. 10000 IU of hCG when at least twofollicles of ≧18 mm in diameter are detected and a serum level of 300pg/ml oestradiol is attained. Alternatively, in embodiments involvingCOH regimes, ovulation may be triggered when the largest follicle hasreached a mean diameter of at least 18 mm, there are at least two otherfollicles with a mean diameter of ≧16 mm (i.e. there are at least 3follicles ≧16 mm and one of these is ≧18 mm) and the oestradiol (E₂)level is within an acceptable range for the number of follicles present(approximately 150 pg/ml/mature follicle). For OI, the ovulationtriggering dose of hCG may be given when there is at least one follicle≧17 mm (and it may be witheld if more than three follicles are ≧15 mm).

hCG has a comparatively long half life in the body. For this reason,when multiple doses are used care must be taken that accumulation doesnot lead to undesirably high levels. It is preferred that serum levelsof hCG not rise substantially above 50 IU/L, preferably not above 25IU/L and most preferably not above about 10 IU/L prior to administrationof the ovulation inducing bolus. If hCG levels rise substantially abovethis level, the result is likely premature luteinisation. Thepharmacokinetics of bolus injections of hCG after intramuscular andsubcutaneous injection have been reported by Mannaerts et al.¹¹

In aspects of the invention where FSH (or an analogue) is used inconjunction with COH techniques or regimens, appropriate doses andadministration regimes will be apparent to a person skilled in the artand any appropriate dose and administration regime may be used. Forexample FSH may be administered daily at a dose of at or about 75 to 250or 75 to 200 IU/day, preferably at or about 150 to 200 IU/day, mostpreferably at or about 150 IU/day. In some patients showing a decreasedresponse to FSH it may be desirable to use doses of up to 600 IU/day. Atypical regimen is as follows: the patient is started on 150 IU FSH/day.After 3 or 4 days, an ultrasound is performed to evaluate developingfollicles. If follicular development is adequate the dose of 150 IUFSH/day may be maintained. If follicular development is inadequate thedose may be increased to 225, 300, 375, 450, 525 or 600 IU FSH/day.Preferably FSH administration continues until the ovulation triggeringdose of hCG is given. Ideally, the cumulative dose of FSH should notexceed 6000 IU/cycle.

The terms “improved rate”, “encouraging”, “aiding”, “increased rates”,etc., as used herein in connection with an effect on implantation orpregnancy include any measurable improvement or increase in frequency ofoccurrence of implantation or pregnancy in an individual patient orgroup of patients treated in accordance with the present invention, forexample when compared with the level or frequency of occurrence ofimplantation or pregnancy in one or more non-treated patients or whencompared to the level or frequency of occurrence of implantation orpregnancy in the same patient observed at an earlier time point (e.g.comparison with a “base line” level). For example, in embodiments wherehCG or LH is used in conjunction with COH, a relevant comparison is tocompare patients treated in accordance with these embodiments to groupsof patients having conventional COH or the same patient havingconventional COH. Preferably the improvement or increase will be astatistically significant one, preferably with a probability value of<0.05. Methods of determining the statistical significance of resultsare well known and documented in the art and any appropriate method maybe used.

The terms “decreased”, “decreasing”, “reduction”, “reducing”, etc. asused herein in connection with an effect on miscarriage, refer to anymeasurable decrease or reduction in the frequency of occurrence ofmiscarriage in an individual patient or group of patients treated inaccordance with the present invention, for example when compared withthe frequency of occurrence of miscarriage in one or more non-treatedpatients or when compared to the level or frequency of occurrence ofmiscarriage in the same patient observed at an earlier time point (e.g.comparison with a “base line” level). For example, in embodiments wherehCG or LH is used in conjunction with COH, a relevant comparison is tocompare patients treated in accordance with these embodiments to groupsof patients having conventional COH or the same patient havingconventional COH. Preferably the decrease will be a statisticallysignificant one, more preferably with a probability value of <0.05. Mostpreferably the uses and methods as described herein in accordance withthe present invention result in the prevention of miscarriages. Thus theprevention of miscarriages is also encompassed by these terms.

The use of hCG (or LH) according to the invention may be useful for anypatient in which it is believed that infertility may be attributed toearly miscarriage or failure to implant, regardless of whether thepatient is receiving other exogenous gonadotropins.

Miscarriage is defined as expulsion of the foetus before it is capableof independent survival. Early miscarriage refers to those miscarriagesthat occur in the first month of foetal development. The methods anduses of the present invention have particular utility in reducing thelevels of early miscarriage.

The use of hCG (and indeed LH) according to the invention in conjunctionwith COH are usually used in conjunction with in vitro fertilisationtechniques. However, it is possible that the uses of hCG (and LH) inconjunction with COH as described herein may also be used in conjunctionwith in vivo fertilisation.

In addition, the use of hCG (and LH) according to the invention may alsobe in conjunction with in vivo fertilisation in patients who are notundergoing COH, for example regimens involving both natural ovulationand ovulation induction regimens, for example using anti-oestrogens oraromatase inhibitors (i.e. regimens not involving the administration ofexogenous gonadotropins). When used in patients receiving no otherexogenous gonadotropins, hCG should be administered starting beforeovulation is expected to occur in any given cycle, preferably startingon or about the 6^(th), 7^(th) or 8^(th) day after menses. A singlebolus may be given (for example at the doses described above for singlebolus administration and in particular 100-500 IU hCG), or it may begiven on a daily basis (for example at the doses described above fordaily administration and in particular at 50-100 IU hCG), or everysecond day (for example at the doses described above and in particularat 50-200 IU hCG), until ovulation takes place either naturally or, ifrequired, by triggering with an ovulation triggering dose of hCG asdescribed above.

In embodiments of the invention where hCG (or LH) are used in patientsnot undergoing COH, such patients may alternatively be undergoing OIusing exogenous gonadotropins (e.g. exogenous FSH). Appropriate dosagesof hCG (or LH) for this use are as described elsewhere herein. Ovulationis triggered at the appropriate time by a high dose of hCG as describedabove.

Thus, as indicated above, it will be appreciated that as well as the useof hCG (or LH) in accordance with the present invention beingadvantageous in conjunction with COH regimes using FSH, hCG (or LH) canalso be used to improve implantation and/or decrease miscarriage ratesof an embryo in a human patient not undergoing a COH regime, but tryingto improve their chances of a successful pregnancy. Such patients (ortheir partners) will generally have experienced fertility problems ofsome kind, i.e. have some level of infertility or sub-fertility.Alternatively, or additionally, the patients may have no obviousfertility problems in terms of ovulation and fertilisation but maydisplay infertility due to a tendency to early miscarriage and/orfailure to implant. Older women e.g. women over 35, where problems withimplantation and higher levels of miscarriage are acknowledged, are alsogood candidates for this treatment. In such patients ovulation mayeither occur naturally or can be induced by an ovulation inductionregime rather than a COH regime, e.g. OI regimes involving aromataseinhibitors, etc. (which stimulate endogenous FSH secretion), orexogenous FSH administration as discussed above. Such OI regimes arestandard and well described in the art.

In such uses not involving COH regimes, the timing of administration ofthe hCG (or LH) is generally calculated from the day of the commencementof menses, although in OI regimes using FSH the timing of administrationcan be calculated from the day of commencing the FSH treatment.Appropriate timings after mensus or after commencing FSH treatment areas discussed above. As discussed above, the hCG (or LH) is administeredstarting before ovulation (which may be either natural ovulation ortriggered by ovulation triggering doses of hCG), during the follicularphase and preferably in the mid-follicular phase of the cycle, e.g. 5 to8 days after menses, e.g. 6, 7 or 8 days after menses, or at least 5 or6 days after beginning FSH treatment, e.g. 5, 6, 7 or 8 days afterbeginning FSH treatment. Thus, it can be seen that although thetreatment regimens in such patients are different from those undergoingCOH using FSH, the preferred timing of administration of the hCG (or LH)is similar, i.e. at or around the mid-follicular phase of the cycle.

When LH is used in conjunction with such methods not involving COH thepreferred timings of administration are as described above for hCG.Preferably the LH is administered on a daily or semi-daily basis atdaily dosages of 125-7000 IU LH or 150-1000 IU LH, more preferably150-700 or 350-700 IU LH. Other doses which may be used are 10 to 200 IULH, 10-150 IU LH or 20-100 IU LH/day. A daily dose of 150 IU LH has beenshown to be particularly effective in the methods of the invention andis preferred. Administration of LH in accordance with the presentinvention has been shown to be particularly effective in women who areat least 35 years of age.

LH, FSH and hCG used in the invention may be formulated foradministration by any convenient route, generally in association with apharmaceutically acceptable carrier, diluent or excipient. Appropriateformulations and routes of administration are well known and documentedin the art for LH, FSH and hCG and any appropriate route and formulationmay be used. Thus, the pharmaceutical compositions of the inventiongenerally contain a pharmaceutically acceptable carrier, diluent orexcipient, together with the appropriate active ingredient.

As mentioned above, patients which may benefit from the methods and usesdescribed herein are any patients which suffer some form of infertilityor subfertility or any patients which wish to reduce the possibility ofmiscarriage and/or to reduce problems associated with improperimplantation, for example patients who have an increased risk ofimproper implantation and/or miscarriage, e.g. patients who are at least35 years old, or patients who have in the past experienced problems withimplantation and/or miscarriage. Suitable patients may have a naturalovulation cycle or may be undergoing OI or COH regimens.

Administration of hCG (or LH) according to the invention is useful inthose patients being treated with GnRH agonists or antagonists. hCG orLH administration according to the invention is particularly useful inconjunction with IVF or IVF/ICSI. The method results in increased ratesof implantation and pregnancies lasting past the first trimester. Theuse of hCG or LH can lead to implantation even in patients who havepreviously demonstrated failure in IVF regimens due to problems notassociated with ovulation.

The use of hCG or LH according to the invention can be particularlyuseful in treating patients with low endogenous LH levels, such aspatients suffering from hypogonadotrophic hypogonadism.

The use of hCG or LH according to the invention can also be used inpatients which have previously exhibited failure to become or remainpregnant using FSH alone, e.g. in standard COH or OI regimens.

Examples of other suitable patient groups are those suffering from PCOD(polycystic ovarian disease), inadequate luteal phase and immunologicalfactors, and patients 35 years old and older (“older patients”).Preferably the patients are not older than 45 years, more preferably notolder than 42 years.

The hCG that is used may be from any source, provided it is notcontaminated with any materials (particularly other gonadotropins) whichwill substantially affect its action. Urinary hCG may be used, althoughit is preferred to use recombinant hCG (rhCG), because of its highpurity. Similar conditions apply to the source of hLH for use in thepresent invention.

Analogues of hCG include all molecules which exert the samephysiological, biochemical or biological effects as hCG, and/or bind tothe same receptors, as hCG. Luteinising hormone (LH) is known to sharesome physiological actions with hCG.

Some analogues of hCG include single chain hCG, in which the C-terminusof the β-subunit is fused to the N-terminus of the α-subunit (Sugaharaet al., PNAS, 92, 1995, 2041-2045). Other examples of analogues are asis disclosed, for example in European patent no. EP 0 322 226 (AppliedResearch Systems), WO 92/22568 (University of Medicine & Dentistry ofNew Jersey), WO 96/05224 (Washington University), WO 90/09800(Washington University), WO 93/06844 (Washington University), WO98/43999 (Washington University), WO 99/25849 (Washington University).

hCG may be detected by any appropriate technique for example usingradioimmunoassay, as described by Vaitukaitis et al.¹², as well as ELISAassays.¹³ The bioactivity of hCG can be measured by any appropriatetechnique, for example, by the mouse Leydig cell bioassay.¹⁴

As mentioned above, the use of LH or an analogue thereof is alsobeneficial during the late stimulatory phase in ART cycles. In COHregimens (i.e. in embodiments when LH is used in conjunction with COH),administration of LH should start before the 10^(th) day after startingFSH administration. Because LH has a relatively short half life,administration on a daily or semi-daily basis is preferred. In aspectsof the invention where LH is used in conjunction with COH, preferredtimings of administration of LH are as described above for hCG. In amost preferred modified COH regimen LH administration is started on orabout the 6^(th) or 7^(th) day after FSH treatment is commenced.Administration of LH should preferably start after the 3^(rd) day aftercommencing FSH treatment. Daily dosages of 125-7000 IU LH or 150-1000 IULH, more preferably 150-700 or 350-700 IU LH may be administered everyday until ovulation is induced. Other doses which may be used are 10 to200 IU LH, 10-150 IU LH or 20-100 IU LH/day. A daily dose of 150 IU LHhas been shown to be particularly effective in the methods of theinvention and is preferred.

In aspects of the invention where LH is used in patients who are notundergoing COH similar daily doses can be used.

Analogues of LH include all molecules which exert the samephysiological, biochemical or biological effects as LH, and/or bind tothe same receptors as LH. hCG is known to share some physiologicalactions with LH. Some examples of analogues of LH are as disclosed, forexample in European patent no. EP 0 322 226 (Applied Research Systems),WO 92/22568 (University of Medicine & Dentistry of New Jersey), WO96/05224 (Washington University), WO 90/09800 (Washington University),WO 93/06844 (Washington University), WO 98/43999 (WashingtonUniversity), WO 99/25849 (Washington University), WO 00/61586 (AkxoNobel).

In embodiments of the invention where FSH is used, it will be understoodby one of skill in the art that FSH may be substituted by a biologicallyactive analogue, or by a compound that stimulates endogenous FSHsecretion. In this latter class are included aromatase inhibitors, andanti-oestrogens such as tamoxifen and clomiphene citrate (CC). Thesecompounds stimulate endogenous FSH secretion by removing the negativefeedback exerted by oestrogen on the hypothalamus (either byantagonising oestrogen receptors, as is the case with CC and tamoxifen,or by greatly decreasing oestrogen concentrations, as is the case witharomatase inhibitors).

A particularly preferred form of FSH for use in conjunction with the useof hCG according to the invention is known as FSH-CTP. This long-actinghuman FSH is described in WO 93/06844, and has a wild type FSH α-subunitand a β-subunit that consists of the wild type FSH β-subunit fused atits carboxyl terminal to the carboxy terminal peptide (CTP) of theβ-subunit of hCG (residues 112-118 to position 145 of the native hCGβsequence). Other types of FSH analogues include, for example singlechain FSH analogues in which the β-subunit is fused to the CTP of hCG,which in turn is fused to FSH α-subunit, as described in WO 96/05224(single chain FSH-CTP).

As for LH and hCG described above, the FSH used in the methods of theinvention can be from any source. Such sources will be well known to aperson skilled in the field of ovulation induction and of COHprocedures. A urinary preparation of FSH may be used, e.g. hMG whichcontains FSH and LH activity at a 1:1 ratio. Preferably recombinant FSHwill be used (rFSH) because of its high purity.

Human menopausal gonadotropin (hMG) has been used to replace FSH duringthe stimulatory phase in ovulation induction and COH for IVF. hMG is arelatively crude hormonal extract from the urine of postmenopausal womenwhich contains both FSH and LH activity (ratio 1:1). Non-proprietary hMGmay contain as little as 2% of the active hormones and consequently asmuch as 98% of the protein content may be urinary contaminants. When hMGis used instead of FSH in the methods of the invention, an hCGsupplementation as described above, may also prove beneficial, forexample for aiding implantation and/or preventing or decreasingmiscarriages. hCG administration should be started before the 10^(th)day after commencing hMG treatment, more preferably before the 9^(th)day after commencing hMG treatment, particularly preferably on or aboutthe 7^(th) or 8^(th) day after commencing hMG treatment. Administrationof hCG should preferably start after the 3^(rd) day after commencing hMGtreatment. Dosages and dosing regimens are as given for use of hCG inconjunction with FSH. A preferred dose is 150 IU/day, more preferably 50or 100 IU hCG/day.

hMG can also be used as a source of hLH in the methods of the invention,i.e. hMG can be used as a source of urinary hLH.

The invention will now be described in more detail in the followingnon-limiting Examples.

EXAMPLES Example 1

Stimulation Protocol

Control group 1: The first day of menses, the patients were submitted tode-sensitisation by daily injections of decapeptyl (0.1 mg). After 14days, a sonographic examination was performed and in the absence ofcysts, stimulation was started with rFSH (150 to 200 IU/day). After 7days, follicular growth was checked by sonography and E₂ bloodconcentration was measured. The patients were examined on a daily basisand ovulation was triggered with 10,000 IU of hCG when at least twofollicles ≧18 mm in diameter were detected and a serum level of 300pg/mL of E₂ was attained. Control group 2: At the time of day 7 incontrol group 1, control group 2 received hMG (150 IU/day) in additionto the rFSH.

Experimental group: At the time of day 7 in the control group, theexperimental group received 50-100 IU of hCG on a daily basis incombination with the rFSH, until ovulation was triggered as above.

The oocytes were fertilised in vitro. Four hours later, they were rinsedand put into culture medium (ISM1). After 20 hrs, fertilisation waschecked and the embryos remained in the same medium until 48 hrs. Theywere then transferred into a second culture medium (ISM2). The 2 bestembryos were then transferred to patients and the remaining werecultured until blastocyst formation was reached (day 5-6).

Results

The results are summarised in Tables 1, 2 and 3. Tables 1 and 2 showresults comparing control group 1 (rFSH alone) with the experimentalgroup. (RFSH+hCG). Table 3 shows results comparing control group 2(rFSH+hMG) with the experimental group (rFSH+hCG).

There was no difference in the lengths of the stimulation periods. E₂levels were increased in the group receiving hCG. The transfer rateswere similar in both groups: 92% with hCG Vs 86% in the control group(p=0.1). Blastocyst formation from the supernumerary embryos was notdifferent between the 2 groups ( 185/411=45% with hCG Vs 292/627=46.5%control, p=0.622). Implantation rates (24.5% hCG Vs 14.6% control,p=0.0134) as well as pregnancy rates per transfer (37.5% hCG Vs 23.6%control, p=0.0246) were increased in the hCG group, yielding a meannumber of 1.9 embryo per transfer.

TABLE 1 hCG low dose in stimulation with rFSH +hCG No hCG No. ofpatients 96 127 Rank (age) 2.2 (32.8 ± 4.1) 1.5 (32.1 ± 4.5) (p =0.1068) Transfers  89 (92%) 109 (86%) Stimulation (days) 11.7 12.3Oocytes recovered 10 9.7 Embryos (m)  6.2 (62%)  5.4 (56%) Implantations43/175 (24.5%) 30/206 (14.6%) (p = 0.0134) Embryo/transfer 1.97 1.89Pregnancies 36/96 (37.5%) 30/127 (23.6%) (p = 0.0246) Blastocysts185/411 (45%) 292/627 (46.5%) (p = 0.6221)

TABLE 2 hCG low dose in stimulation with rFSH +hCG No hCG TotalPregnancies 109 230 339 Ectopics  2  6  8 (2.4%) (p = 0.95) Miscarriages 8 (7.3%)  36 (15.6%)  44 (13%) (p = 0.03) Total miscarriages +  10(9.17)  42 (18.3)   52 (15.7) (p = 0.03) ectopics

TABLE 3 hCG low dose in stimulation with rFSH Vs hMG FSH +hCG hMGPregnancies 109 76 Ectopics 2  2 Miscarriages 8 13  (p = 0.049) Totalmiscarriages + ectopics 10 15  (p = 0.049)

Example 2

The following study compared the pregnancy outcome of patients treatedin COH using FSH alone, with those treated using FSH plus LH starting onday 6.

Following a negative quantitative serum pregnancy test, eligibleparticipants underwent pituitary desensitisation using the gonadotrophinreleasing-hormone agonist (GnRH-a), Lupron®, starting 7 to 8 days afterestimated ovulation at a dose of 0.5 mg daily until desensitisation wasestablished by a serum estradiol (E2) level of <75 pg/mL. At that time,treatment with recombinant human FSH at a starting dose of 225 IUsubcutaneous per day was initiated. This dose continued for the first 5treatment days at which time the dose could be increased by 75 to 150IU/day every 2 to 3 days if the patient's ovarian response was judged tobe slow. The maximum dose of FSH allowed was 450 IU/day and the maximumcumulative dose was not to exceed 6,000 IU per cycle. For those patientsrandomised to receive FSH plus r-hLH, r-hLH treatment was initiated at adose of 150 IU per day on stimulation Day 6. The dose of r-hLH was notto be modified. After achieving down-regulation, Lupron treatmentcontinued throughout the stimulation cycle at a dose of 0.25 mg dailyuntil administration of hCG. Estradiol levels and ultrasoundmeasurements were assessed throughout the stimulation cycle to determinepatient response to treatment. The administration of FSH or FSH plusr-hLH continued daily until follicular development was consideredadequate. The criteria for hCG administration (for ovulation triggering)was met when the largest follicle reached a mean diameter ≧18 mm and atleast two other follicles had a mean diameter of ≧16 mm. The patientsalso needed an E2 level within the investigator's acceptable range forthe number of follicles present (approximately 150 pg/ml/maturefollicle). At that time, the patient received a single IM injection of10,000 USP units of hCG for the final stage of follicular maturation.Oocytes were recovered vaginally under ultrasound monitoring 34 to 36hours after the administration of hCG in accordance with the usualpractice of the investigational site. Intracytoplasmic sperm injection(ICSI) was then performed according to the standard procedures at eachsite. Up to 3 embryos could be replaced 2 to 3 days after ovum pick-up.Luteal phase support with natural progesterone in oil was provided forall patients who received hCG starting on the evening of oocyteretrieval and continuing for a minimum of 7 days; the investigator couldthen choose to use progesterone suppositories. The total duration ofluteal phase support was at the discretion of the investigator.

A blood sample for serum β-hCG level was collected on day 15 to 17 afterhCG injection for all patients who underwent embryo transfer. If theresults were positive (biochemical pregnancy), the test was repeated 2to 7 days later.

A post-treatment visit including general physical examination andclinical laboratory testing was conducted in all patients 15 to 17 daysfollowing hCG administration (when the patient returned for pregnancytesting) or within one week of onset of menses. For all patients whobecame pregnant, an ultrasound scan was performed 35 to 42 days afterhCG administration and the number of fetal sacs and fetal heart activitywas recorded (“clinical pregnancy”). If clinical pregnancy occurred,women were followed to determine pregnancy outcome.

The data were analysed dividing the patients into those aged 35 yearsand older (“older patients”), and those aged less than 35 years. Theclinical pregnancy data are shown in Table 4. The treatment of FSH+LH isclearly better in pregnancy outcome in patients aged 35 years and older(48.8% with FSH+LH VS 21.6% with FSH alone).

TABLE 4 Pregnancy data for patients treated with FSH alone versuspatients treated with FSH + LH (LH starting on day 6) FSH + LH FSH <35≧35 Overall <35 ≧35 Overall Number of patients (N) 103 41 144 107 37 144Clinical 46 20 66 48 8 56 pregnancies (44.7%) (48.8%) (45.8%) (44.9%)(21.6%) (38.9%) (%)

REFERENCES

-   ¹ Healy et al.; Lancet 343 1994; 1539-1544-   ² for example, a conventional technique is described in EP 0 170 502    (Serono Laboratories, Inc.)-   ³ Filicori, M.; J. Clin. Endocrinol. Metab. 81 1996; 2413-6-   ⁴ Filicori, M. et al; Fertil. Steril. 65 1996; 387-93-   ⁵ Hillier et al; Horm. Res. 43 1995; 216-223-   ⁶ Esposito et al.; Fertility & Sterility 75 2001; 519-524-   ⁷ The European Recombinant Human LH Study Group; J. Clin.    Endocrinol. Metab. 83 1998; 1507-1514-   ⁸ Messinis et al.; Fertility & Sterility 50-1988; 31-35-   ⁹ Lei et al.; J. Clin. Endocrinol. Metab. 75 1992; 651-659-   ¹⁰ Bennett et al.; Pharmacol. Rev. 30 1979; 247-292-   ¹¹ Mannaerts et al.; Human Reproduction 13 1998; 1461-1464-   ¹² Vaitukaitis et al.; Am. J. Obstet. Gynecol. 113 1972; 751; Clin.    Chem. 31 1985; 1749-   ¹³ Tyrey et al.; Obstet. Gynecol. Clin. North Am. 15 1988; 457-   ¹⁴ Robertson, W. R. and Binden, S. P; The in vitro bioassay of    peptide hormones. In Hutton, J. C. and Siddle, K. (eds), Peptide    Hormones; a Practical Approach. IRL Press, Oxford (1990).

1. A method for aiding/encouraging implantation and/or decreasingmiscarriage rates of an embryo in a human patient, comprisingadministering human luteinising hormone (hLH) during controlled ovarianhyperstimulation (COH) in a human patient in need thereof, whilecontinuing administration of FSH, wherein the hLH is administeredstarting between the 3^(rd) and 10^(th) day after commencing treatmentwith FSH, to aid/encourage implantation and/or decrease miscarriagerates of an embryo in the human patient.
 2. The method according toclaim 1, wherein the hLH is administered at a dosage of 125-7000 IUhLH/day.
 3. The method according to claim 1, wherein the hLH isadministered starting between the 3^(rd) and 8^(th) day after commencingtreatment with FSH.
 4. The method according to claim 3, wherein thehuman patient is at least 35 years of age.
 5. The method according toclaim 1, wherein the hLH is recombinant hLH.
 6. The method according toclaim 1, wherein the hLH is urinary hLH.